Concentration of protein and/or peptides samples

ABSTRACT

The invention relates to a device for protein and/or peptide concentration, which device comprises electroconcentration means ( 23 ); at least two electrodes having a positive ( 7 ) and a negative charge ( 35 ), respectively; and protein and/or peptide capture means ( 17 ); wherein said electroconcentration means ( 23 ) comprises a funnel shaped cavity and at least one electrode is located on each side of the electroconcentration means. The invention also relates to a method for concentrating a protein and/or a peptide in a sample, which method can be performed in a device according to the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/EP02/10118 filed Sep. 10,2002, published on Mar. 27, 2003 as WO03/025578, and to foreignapplication number 0122200.9 filed in Great Britain on Sep. 14, 2001,the entire disclosures of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a device useful for the production,immobilisation and subsequent handling and chemical modification ofsmall amounts of peptides prior to quantification, identification andcharacterisation thereof. The invention also encompasses a method forsuch production, immobilisation and subsequent modification.

BACKGROUND

Protein identification has been revolutionised by the introduction ofmethods to identify proteins in databases using mass spectral data,either based on peptide masses from protein digests or fragmentationspectra from individual peptides. Interestingly, the main problem thatis encountered in high sensitivity protein/peptide analysis andidentification is usually not related to the sensitivity of theanalysing device (usually a mass spectrometer, which can sequencepeptides at the attomole level), but the generation and handling ofpeptides by digestion of the protein.

In the prior art, the target protein has usually been isolated bytwo-dimensional gel electrophoresis (2D-PAGE) or affinity chromatographyand recovered in a fairly large volume of solution, such as about 20 μlfor a spot from a two-dimensional electrophoresis gel. Thus, proteinshave been recovered in a very diluted state. For example, if a microgramof material is available of a 50,000 MW protein, (20 pmol) theconcentration is 1 μM. This is slightly below the Km of most proteases,and even though digestion can still take place, it will be a very slowprocess. Further, if only a nanogram is available, i.e. 20 femtomoleswhich is well within the sensitivity range of modern mass spectrometersusing nanospray ionisation, digestion will not occur to any significantextent since then the protein solution is too dilute.

A further drawback of the prior art devices is that the container walls,which are usually of plastics or glass, absorb peptides and proteinsvery easily and large losses will occur within an hour or so of theprotein/peptide solution coming into contact with the container.Furthermore, subsequent handling steps such as pipetting or being loadedinto a chromatography system all involve contact with large surfaceareas and dramatically increasing sample losses.

For a general review of the above-mentioned methods, see e.g.Staudenmann, W., Dainese Hatt, P., Hoving, S., Lehmann, A., Kertesz, M.,and James, P. (1998) Electrophoresis, 19, 901–908. “Sample handling forproteome analysis.”

SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide a device and a methodthat improves handling, such as concentration, digestion and/or chemicalmodification, of small amounts of peptide and/or protein. Another objectis to provide such a device and method wherein the peptide and/orprotein immobilises directly on a surface from which a subsequentanalysis can be performed by any suitable method. A further object ofthe present invention is to provide a device and a method for the abovementioned handling that allows it to take place with reduced losses ofpeptide and/or protein due to adsorption to container walls, and whichthereby allows a prolonged maintenance of peptide/protein sample in thedevice.

The objects of the invention can be achieved by the device and method asdisclosed in the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 a) shows a schematic representation of a section though an arrayof devices according to the present invention for carrying outsimultaneous concentration-digestion of a protein sample;

FIG. 1 b shows a view from above of the array of FIG. 1 a);

FIG. 2 a) shows a schematic representation of a section through an arrayof flow through chemical cells for flow-through high-efficiency chemicalmodifications according to the present invention;

FIG. 2 b) shows a partial section from the side of a target slide andmask;

FIG. 2 c) shows schematically a view from above of a plate with 100loading positions for stations of the type shown in FIG. 2 a); and

FIG. 2 d) shows schematically a view from above of a plate with 50loading positions.

DEFINITIONS

In the present specification, the term “capture” means to non-covalentlybind e.g. a peptide to e.g. a support.

The term “protein and/or peptide” is understood to include any chain ofamino acids or modified forms thereof.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a device for protein and/orpeptide concentration in a sample, which device comprises:

electroconcentration means comprising a funnel shaped cavity with a wideend and a narrow end; at least two electrodes, one electrode beingpositioned near to said wide end and one electrode being positionednearer to said narrow end; and one or more protein and/or peptidecapture means; wherein said capture means is located between said narrowend and said one electrode positioned near said narrow end.In the preferred embodiment, the present device is presented as anassembly held together by a seal. During use, the whole device ispreferably held within a pressurised container at around 2–3 bar toprevent the formation of bubbles which otherwise might form duringelectrophoresis from blocking the passages and stopping the currentflow.

The sample can be presented to the device in an electroelution chamberwhich chamber is present in an electroelution bath. The electroelutionchamber can be fixed, e.g. by clamps, to an analysis target, such as aMALDI target, for subsequent analysis.

The types of material used to make device may vary according to the modeof analysis. Thus, the electroelution bath can be made from any materialthat shows a sufficiently low protein absorbance and which is alsopreferably easy to machine, such as Plexiglas. The same criteria formaterial properties can also be applied to the electroconcentrationmeans. Electrodes, clamps, and analysis target can be made from achemically inert metal such as stainless steel or a metal coated with anoble metal such as gold. The seal can be any flexible material such assilicone rubber tubing. The capture means includes a trapping materialsuch as C18 silica embedded in Teflon discs, since these aremechanically stable and very chemical resistant. This allows one tocarry out chemical reactions such as chemical digestions in the gasphase with reagents such as cyanogen bromide, pentafluoropropionic acidand S-ethyltrifluoroacetate. However, other trapping materials can beused and are easily selected by the person skilled in this fieldaccording to the method of analysis to be used subsequently. In aspecific embodiment, the analysis target, which is constituted of piecesof metal, is made of chemically resistant metal when the sample is goingto be analysed by MALDI-TOF mass spectrometry, since one has to fix themembrane at a defined potential in the instrument. Otherwise othermaterials may be used depending on the analysis method to be used. Inone embodiment, the analysis target contains a heating element so thedigest temperature can be maintained at about 37° C. In an alternativeembodiment, the temperature is controlled with an external heatingsource. An advantageous feature of the invention is that there may be‘teeth’, or a raised edge, around each hole holding the capture means,providing a minimal or even no cross flow of reagents or samples. Otherelution formats can be used, e.g. a dual elution chamber.

In one embodiment of carrying out the present invention, the protein(s)and/or peptides are equilibrated with a detergent or solubilising agentsuch as sodium dodecyl sulphate or urea, until they are fully insolution. However, the protein(s) and/or peptides can alternatively bepresent either in a gel slice, such as a 2D gel spot, or attachednon-covalently to any other carrier. The protein-containing sample isthen placed in the funnel of the electroconcentration funnel block.

The volume of the funnel shaped cavity can be designed to accommodateany volume but a volume of between about 0.5–10 ml, which corresponds tothe range of commonly used sample volumes, is preferably used. Thefunnel inlet size is not critical, however the size of the narrow,outlet end of the funnel is important for two reasons. Firstly, it isthe volume of the outlet end part of the funnel shaped cavity thatdefines the final concentration of the material undergoing digestion.Since the Km of most proteolytic enzymes is in the range 5–50 μM,digestion of proteins will only be effective in the pmol/μl range. Forexample, the narrow end of the funnel shaped cavity should have a volumeof less than about 100 nl if protein amounts are in the hundreds offemtomoles region As the person skilled in this field realises, itshould be proportionately smaller if the amount to be digested is less.Secondly, the diameter of the outlet hole defines the area ofpeptide/protein capture, which is the area that will be subsequentlyused for analysis. The smaller the area, the higher the concentration ofdigested or concentrated material, and hence the higher the sensitivityin applications such as matrix-assisted laser desorption and ionisation(MALDI) mass spectrometry (MS), fluorimetry (for fluorine labelledmaterial) or various antibody based detection methods. The minimum sizeis defined by detection method, for example with MALDI-MS, the size ofthe laser spot. Practical experience defines the minimum useful sizei.e. the narrowest point of the funnel next to the MS cut-off membrane,in this configuration to be around 30 μm. Thus, in one embodimentdescribed below, the dimension of the outlet of the narrow end of thefunnel shaped cavity of the device has been adapted to the envisagedmethod of analysis.

Thus, the present invention discloses a device that allows theconcentration, digestion and/or chemical modification of very smallamounts of protein and/or peptide and immobilises them directly on asurface from which they can be subsequently analysed by the method ofchoice.

In accordance with the present invention, concentration of an isolatedprotein or proteins can be obtained down to a final volume of around20–100 nl or lower, as compared to the prior art, where digestion hasusually been performed in the gel spot which is around 20 μl or on aelectroblotting support which requires around 5 μl.

In one embodiment of the present device, the capture means is in theform of an immobilisation support capable of capturing positively and/ornegatively charged peptides. In other embodiments, specificimmobilisation supports can be created by using antibodies, affibodiesetc. Alternatively, specific binding properties of proteins can be usedand ion exchange or specific immobilised metal ion chromatography (IMAC)supports can be used to capture acidic, basic or phosphopeptides.

In one advantageous embodiment of the present device, the funnel shapedcavity of the electroconcentration means is in a position wherein saidcavity has its longitudinal axis in an essentially vertical position,with the wide end above the narrow end.

The present device can be operated in two different modes. The first ofthese embodiments is advantageously used when proteolysis is carried outusing an enzyme or when peptides are to be extracted from a mixturecomprising higher molecular weight molecules. The device according tothe invention then further comprises a semi-permeable restrictionmembrane or a size cut-off membrane positioned in the peptide's pathsuch that peptides must pass through the membrane before they can reachthe capture means. The membrane can be made of cellulose acetate and tomake the pores smaller and stabilise the structure both mechanically andchemically, it can be treated e.g. with acetic anhydride. Alternativesinclude any materials that can be made with a defined pore size rangesuch as silica glass or a polymer such as acrylamide and these can bemade as an integral part of the device rather than just as a membrane.When using this embodiment, a sample is placed in the device, an enzyme,such as a protease, is added and the sample is co-concentrated. Theproteins remain in solution and can now be efficiently digested by theenzyme since they are now very much more concentrated. A membrane(available from a number of commercial sources, such as Spectraporeinc.) with a molecular cut-off weight e.g. of around 3000 Dalton willthen be placed between the protein solution and the capture means. Thus,only smaller peptides which are amenable to MS/MS analysis are trappedin the capture means and any large molecules such as polymers like PEGor big proteins which interfere with the MS analysis will be removed bythe cut-off membrane. The proteins concentrate above the cut-offmembrane but cannot pass through it, however the peptides being releasedduring digestion can pass through it and are subsequently immobilised ona capture membrane prior to subsequent handling.

In the second embodiment, the present device can be operated without amembrane. This is especially advantageous when the captured protein orpeptide is to be digested with a chemical and the concentration factoris determined by the diameter of the end of the concentration funnel.Suitable chemicals for this purpose are e.g. cyanogen bromide,pentafluoropropionic acid or S-ethyltrifluoroacetate. For example, inorder to digest femtomole amounts of material the protein must beconcentrated from the usual 1 fmol/μl range up to the pmol/μl range.

Thus, in one embodiment, the present device is especially adapted fortagged proteins. In this embodiment, the present device comprises twocapture means separated from each other, one of which is preceded by amask allowing unmodified peptides to pass through and the other one ofwhich is preceded by a mask allowing modified peptides to pass throughand adapted to generate a peptide fingerprint by ladder chemistry. Inthis embodiment, the tag is the sequence generated by ladder chemistry,which is a well known concept to those skilled in this field. Thechemistry preferably used herein is thioacetylthioester degradationsimilar to the well known Edman degradation. More detailed, it is setupto be inefficient so 20% or so of the N-terminal amino acid of a peptideis removed. This is repeated ×3 to give some 40% intact peptide 1, 30%peptide 1—1 amino acid, 20% with two amino acids removed and 10% withone amino acid removed. By the mass differences between the peaks in theMS one can read a sequence. For example, if the parent peptide mass1000, −1aa mass 943, −2aa 830, −3aa 701, then the sequence tag is 1000,glycine, leucine, glutamic acid.

In an advantageous embodiment, the capture means, e.g. the membranesupport, can subsequently be used as a flow-through reactor to allowhigh efficiency chemical modifications of very small amounts of materialand/or can be used directly for analysis by mass spectrometry,functional activity assays or fluorescence detection for example. Thedevice can be operated in parallel allowing large numbers of proteins tobe concentrated, digested and immobilised on a membrane for subsequentmodification and/or analysis. Accordingly, the device according to thepresent invention is especially suitable for automated procedures, whereit will enable substantial savings in process time and costs, ascompared to the prior art methods. Thus, in one embodiment of thepresent device, the capture means are adapted for use in MALDI-MS.

In the experimental part below, one illustrative embodiment of a deviceaccording to the present invention will be described in more detail withreference to the drawings. As the person skilled in this field willrealise, the present device can however be constructed in alternativefashions for example, as a silicon chip, as arrays, as single units, asdisposable units, as reusable units, etc. In all cases, the principleremains the same, namely, the concentration of the protein in the liquidphase to allow digestion or capture on an immobilising surface forsubsequent analysis.

A second aspect of the present invention is a method for concentrating aprotein and/or a peptide in a sample, comprising the steps of

-   -   (a) Providing a sample which comprises proteins and/or peptides        and a digestive agent in an electrophoresis device, wherein the        electroelution bath is present in an essentially funnel shaped        cavity;    -   (b) Applying a voltage between at least two electrodes located        on each side of said electroelution bath to pass peptides        towards a capture means located between the narrow end of said        funnel shaped cavity and the electrode positioned nearer said        narrow end;    -   (c) Changing the direction of the voltage at least once to        provide oscillations enabling both positively charged and        negatively charged peptides to contact the capture means; and,    -   (d) Collecting concentrated peptides from the capture means.

The voltage causes the proteins and peptides to move in a certaindirection and the speed of movement is inversely proportional to thesize so proteins remain essentially where they are since they arenegatively charged due to detergent such as SDS present in the sample.Once digested, the resulting peptides can be positively or negativelycharged. Hence, without reversal of the voltage occasionally, one setwill be lost, since then they move away from the capture membrane.Usually, the actual digestion during the first voltage will last for alonger time, such as a couple of hours, as compared to the subsequentchanges of the voltage direction, which changes can be made more often,e.g. every 10 minutes. However, the exact intervals and reaction timesare easily determined by the skilled in this field for each desiredconcentration procedure.

In one embodiment of the present method, the digestive agent is anenzyme, preferably a protease (such as trypsin, V8 protease, LysC, AspNetc) or a glycosidase to release O- and N-linked saccharides for captureon a hydrophilic support prior to subsequent analysis of theglyco-portion.

In another embodiment of the present invention, the method above isadapted to comprise the further step of separating larger sizemolecules, such as proteins, from smaller size molecules, such aspeptides, by introducing a size cut-off membrane between the narrow endof the funnel shaped cavity and the capture means. The membrane is thereto prevent proteins from reaching the capture means during the digestionand also at the end of the digestion, when the sample is convenientlyallowed to flow downwards through the device. Thus, it prevents anylarge fragments from coming onto the membrane. In this context it isunderstood that the term “large” refers to fragments of an undesiredsize, and that the cut-off value of the membrane is selected dependingon the desired size of peptides.

In one embodiment of the present method, in step (b), the sample isdivided after the funnel shaped cavity into two or more parts and eachpart is passed towards a separate capture means, one capture means beingpreceded by a mask allowing unmodified peptides to pass through and theother one being preceded by a different mask allowing modified peptidesto pass through, and in step (d), unmodified peptides are collected fromone capture means and tagged peptides providing a peptide fingerprintare collected from another capture means. Thus, tagging is allowed, asdiscussed above in relation to the device. Since each digest generates aset of peptide masses, known as the peptide fingerprint, one can usethis to identify a protein in a database that generates a similartheoretical set of peptides when digested by the same enzyme. This isknown as ‘peptide mass fingerprinting’. However, in the prior art, thishas often shown to be not enough for a reliable identification. Thus,the present invention now allows one to generate a peptide fingerprintas well as a small sequence of three amino acids per peptide. Thisallows proteins to be identified at a much greater confidence level andfar more efficiently than previously. For details to this end, thetagging technology has been described: for an algorithm foridentification using sequence tags and the method to generate them usingexopeptidases, see Korostensky, C., Staudenmann, W., Dainese, P.,Hoving, S., Gonnet, G., and James, P. (1998) Electrophoresis, 19,1933–1940. “An algorithm for the identification of proteins in sequencedatabases using peptides with ragged N- or C-termini generated bysequential endo- and exopeptidase digestions”, and for a description ofthe method of generating the tags chemically, see Hoving, S., Münchbach,M., Quadroni, M., Staudenmann, W., and James, P. (2000) Anal. Chem. 72,1006–1014. “Multiple N-terminal tag generation from unseparated proteindigests using a novel thioester based degradation reaction”.

In a specific embodiment, the peptide fingerprint is generated bythioacetylthioester degradation. This can also be carried out by Edmandegradation using phenylisothiocyanate or any other suitable reagentwith an isothiocyanate moiety.

In an additional embodiment of the method, the proteins and/or peptidesto be concentrated are present in a gel or in a solution. Alternatively,they can be non-covalently bonded to a support. The enzyme can e.g. beadded to the sample in the electroelution bath before applying thevoltage to digest proteins into peptides or can be rehydrated to the gelpiece.

One specific embodiment of the present method is a method according tothe invention which is suitable for analysis of a protein and/or peptidesample and which comprises the further step of inserting the capturemeans obtained in step (d) into a detection apparatus, such as aMALDI-MS. In this context, phosphorylation and glycopeptide eliminationanalysis can advantageously be used. For more details to this end, amethod for phosphorylation analysis that is done in a test tube has beendescribed, see Quadroni, Q. (2000) Principles and Practice. ProteomeResearch: Mass spectrometry. Specific detection of analysis ofphosphorylated peptides by mass spectrometry, pp 187–206. Wiley-SpringerVerlag. Ed. James, P.

An advantageous embodiment of the present method uses a device accordingto the invention as described above.

Thus, in summary, in one illustrative embodiment of the present method,a sample comprising proteins is first equilibrated as described aboveand enzyme, such as protease or a digestive chemical is added. Theelectrophoresis is started after said addition. The sample is focusedonto a low molecular weight cut-off membrane as discussed above and thenthe polarity of the voltage is reversed once or several times, e.g.every five or ten minutes for one hour. The fragments which are smallenough to pass the first membrane migrate very quickly (since they aresmall) onto the C18 membrane where they are immobilised. The buffers arepreferably removed from the bath and then water is passed through fromtop to bottom through the immobilising support to remove undesiredsalts. In one embodiment the present method also comprises a step ofanalysis, wherein the device is then disassembled and inserted into themeasuring device of choice. For example, if the sample is to be measuredby MALDI, the support is inverted and about 1–2 μl of MALDI matrix suchas α-cyano-4-hydroxy-cinnamic acid, 10 mg/ml in 80% acetonitrile, 1.25%TFA in water is added and allowed to flow into the membrane. The supportis then suitable for being placed into the MALDI-MS. In an alternativeembodiment, the support can be used as a nano-electrospray tip. Thebottom end of each channel leading out of the MALDI target can be madein a sharp conical form with about 10 μm diameter outlet. In thiscontext, it is understood that the term “sharp conical form” means aconical shape with a hole in the centre, wherein the walls of the exitpoint around the hole are very narrow/sharp i.e the internal angle ofthe cone is preferably less than, 45° and most preferably 30° or less. Ahigh-pressure liquid chromatography line can be clamped across thetarget and a gradient ran out to allow HPLC-MS measurements. Anotherembodiment utilises a double trapping layer with C18 particles first andthen a strong cation exchanger. The sample can be measured using MALDIfirst and then the matrix washed away by any suitable solution leavingthe peptides bound to the strong cation exchanger and eluting thepeptides with a suitable gradient of e.g. acetonitrile in water with TFAor formic acid.

In summary, the present invention describes a novel device and method,which compared to the prior art gel-based concentration system for theconcentration of proteins from 2D gels includes the followingadvantages:

According to the present invention, there is no need to stain thesample. In the prior art, the protein must be stained to be visualisedand this causes losses during fixing, as well as time consuming stainingand destaining steps, especially with small proteins. Even thoughprotein could be collected without staining by reference to its positionrelative to a dye front, this is unreliable and leads to larger volumesof gel being cut out in order to ensure complete recovery of theproteins.

Further, according to the present invention, proteins are easilyaccessible to proteases and digestive chemicals, as compared to theprior art methods, where protein trapped in the polymer matrix canremain inaccessible to proteases.

In the prior art methods, the buffers and detergents present disturbHPLC and MS analysis and must be removed to allow high sensitivityanalysis causing further losses. There is no such need according to thepresent invention.

Finally, subsequent pipetting and transfer steps as well concentrationby lyophilisation required in the prior art cause further sample losses.Another sample loss is especially critical, namely the loss ofphosphopeptides onto metal surfaces, such as inside of metal HPLCcolumns, frits at the beginning and end of columns, injection valvesetc. Such losses are also avoided according to the present invention.

The present invention will now be described by way of examples, whichare provided for illustrative purposes only and which should not beconstrued as limiting the invention as defined by the appended claims.All references given below and elsewhere in the present application arehereby included herein by reference.

EXPERIMENTAL PART Example 1 Device for Digestion

A device according to the invention, which is especially adapted for usewith MALDI, is described below with reference to FIGS. 1 a) and 1 b).FIG. 1 a) shows schematically a section through part of an array 1 ofsubstantially identical electroconcentration cells 3 formed in asubstrate 5 and FIG. 1 b) shows a view from above of the section of FIG.1 a). The array may comprise any suitable number of cells, preferablyarranged in a grid pattern comprising a number of rows and columns, forexample 8 rows and 12 columns or 10 rows and 10 columns, etc. Theconstruction of one such cell shown in FIG. 1 a) and FIG. 1 b) will nowbe described. Cell 3 is preferably formed as a regularly shaped cavity3, in this example a cube, formed in said substrate 5. A firstelectroconcentration electrode 7, connected to a power supply (notshown) is provided at the lower part of cavity 3. A spacer means in theform of a wall 9 or the like, projecting a small distance towards thecentre of cavity 3 and extending a distance from the base of the cavity3 that is sufficient to contain first electrode 7, supports a MALDItarget slide 12. MALDI target slide 12 comprises first and second thinsquare sheets 11,19 of conducting material such as stainless steel,aluminium or the like, which are placed with second upper sheet 19 ontop of, and in contact with first, lower sheet 11. Sheets 11, 19 arejoined together by any suitable means such as gluing, welding, brazing,crimping, folding, riveting, being bolted together, etc. Sheets 11, 19have sides which are as long as or slightly smaller than the lengths ofthe inner walls of cavity 3 such that MALDI target slide 12 can fitinside cavity 3. Preferably the dimensions of MALDI target sheets 11,19, wall 9 and cavity 3 are adapted so that when MALDI target slide 12is positioned on wall 9 no fluid can flow past the outer edges of theMALDI target slide 12. Seals, not shown, may also be provided asnecessary to accomplish this. MALDI target lower sheet 11 has a centralthough hole 13 which opens into a cylindrical cut-out 15 on the uppersurface of lower sheet 11. A capture means 17 in the shape of a dischaving a diameter less than the diameter of cut-out 15 and a height lessthan or equal to the height of cut-out 15 is positioned in cut-out 15.Capture means can be in the form of a semi-permeable membrane, e.g.comprising C-18 silica particles in a Teflon™ membrane, which cancapture molecules of interest. Second, upper sheet 19 has a centralthrough hole 21 that is arranged to be substantially concentric with thethrough hole 13 of first sheet 11. An electroconcentration block 23 madeof a chemically inert, insulating material such as Plexiglas™ ispositioned on top of, and in sealing contact with second, upper sheet19. Block 23 has sides, which are as long as or slightly smaller thanthe lengths of the inner walls of cavity 3 such that block 23 can fitinside cavity 3. The height of block 23 is sufficient to contain adigestion funnel and membrane as described below. The bottom surface ofblock 23 which is in contact with the upper surface of upper sheet 19 ofthe MALDI target slide 12, has a cylindrical central cut-out 25 that issubstantially concentric with the cut-out 15 of lower sheet 11. Cut-out25 contains a weight cut-off membrane, e.g. a 3000 Da molecular weightcut-off membrane 27. Block 23 further comprises a channel 29,substantially concentric with though holes 13, 21. Channel 29 extendsfrom cut-out 25 and forms the narrow spout of an inverted-cone-shapedelectroconcentration funnel 31 formed in block 23. The upper, wider endof funnel 31 is sufficiently wide enough to receive a gel sample 33, andwill in general be of a size of about 1–10 mm in diameter, and funnel 31tapers down to the diameter of channel 29. A second electrode 35 ispositioned above block 23 and is immersed in bath fluid 37. Bath fluidfills the cell 3 so that first electrode 7 is in electrical contact withsecond electrode 35.

The whole device is held within a pressurised container at around 2–3bar to prevent bubble formation during electrophoresis from blocking thepassages and stopping the current flow.

The weight cut-off membrane 27 is preferably made of cellulose acetate,which has been acetic anhydride treated. The trapping material for thecapture means 17 is C18 silica embedded in Teflon discs (known as“Empore™”), which allows one to carry out chemical reactions such aschemical digestions in the gas phase with reagents such as cyanogenbromide, pentafluoropropionic acid and S-ethyltrifluoroacetate. TheMALDI target (i.e. the sheets 11, 19 between which the Empore membraneis clamped) is made of chemically resistant metal since in MALDI-TOFmass spectrometry one has to fix the membrane at a defined potential inthe instrument. The MALDI target can contain a heating element (notshown) so the digest temperature can be maintained at a desiredtemperature, e.g. 37° C. The molecular weight cut-off membrane 27 andthe C18-Teflon material are each one membrane, which covers the entiretyof the target. There is no cross flow of reagents or samples since‘teeth’ or a raised edge around each hole holds the membranes.

The electrophoresis is started after the addition of proteolytic enzyme.The protein and enzyme are focused onto the low molecular weight cut-offmembrane by using a potential difference between the electrodes of 300Vfor 2 hours, and then the polarity of the voltage is reversed every fiveminutes for one hour. The fragments which are small enough to pass thefirst membrane 27 migrate very quickly (since they are small) onto theC18 membrane 17 where they are immobilised. The buffers are removed fromthe bath and then water is passed through from top to bottom through theimmobilising support to remove salts. The device is then disassembled,the MALDI target is inverted and 1–2 μl of MALDI matrix in the form ofα-cyano-4-hydroxy-cinnamic acid, 10 mg/ml in 80% acetonitrile, 1.25% TFAin water is added and allowed to flow into the membrane 17. The targetis then placed into a MALDI-MS. In this embodiment, preferably thebottom end of each though hole leading out of the MALDI target is madewith a sharp conical form with a 10 μm diameter exit (for reasons ofclarity of illustration, these holes are not drawn to scale in thefigures). A high-pressure liquid chromatography line is clamped acrossthe target and a gradient ran out to allow HPLC-MS measurements.

Example 2 Concentration of Peptides

Conditions for sample solubilisation from gels: The protein cut from thegel contains 0.1% SDS if cut from a non-fixed gel (covalent fluorescentstaining or reversed staining) or 0% SDS if fixed and washed (silver,Coomassie etc stains). Stained proteins are solubilised by leaving thegel overnight in 3% SDS. The final concentration in the gel for bothsample types is between 0.001–0.03%. This helps saturate the surfaces ofthe device lowering absorption and increases the mobility of theprotein.

Experimental description: Ten proteins (horse heart cytochrome c,recombinant whale myoglobin, calmodulin, bovine serum albumin, and sixsubunits (30S subunits S1, S2, S4, S8, S11, and S16) of the small 30Sribosome from Escherichia coli) were digested at varying concentrationseither in solution in an Eppendorff tube or in the device. Thedigestions were carried out in all cases with 0.1 μg of trypsin in 100mM ammonium bicarbonate and 0.001% SDS at 37° C. in a volume of 10 μl inthe Eppendorff or added to the device. A voltage of 300V was applied for2 hours after loading and the device was maintained at 37° C. After twohours the polarity of the voltage was reversed every 10 minutes for thenext four hours. The solution digestion was stopped after six hours bypassing the solution through an Empore C18 membrane. The peptides wereeluted using 5 μl of 60% acetonitrile, 0.1% trifluoroacetic acid inwater and the entire 5 μl was loaded onto the MALDI target by multiplespotting and drying onto a surface precoated with matrix and the massspectrum was measured. The digestion in the device was stopped byremoving the voltage and bringing the solution to flow through theimmobilising support in the device. The device was disassembled andmatrix in 80% acetonitrile was spotted onto the immobilising support onthe side that was not in contact with the protein solution.

Summary of results averaged obtained from the ten proteins:

Average number of peptides in spectra Protein (from 800–3000 m/z)(excluding tryptic autoproteolytic concentration fragments) obtainedfrom the ten proteins (pmol/μl) In solution In device 100 24 23 10 24 241 20 22 0.1 10 22 0.01 3 23 0.001 0 17

Example 3 Ribosomal Protein Isolation

Escherichia coli MC4100 (F-araD139 D(argF-lac) U169 rpsL150 relA1 deoC1ptsF25 rpsR flbB5301) was obtained from the laboratory collection.Bacteria were cultivated in a sulphur-free, synthetic glucose-saltsmedium as previously described, with the addition of 500 μM inorganicsulphate. The cultures were grown aerobically on a rotary shaker (180rpm) at 37° C., and growth was monitored spectrophotometrically at 650nm. Cells were harvested in the mid-exponential phase (A₆₅₀=0.5) bycentrifugation (7,000×g, 10 min, 4° C.) and washed with 50 mM Tris/HCl,pH 7.0. They were then resuspended in the same buffer (0.8 g wet massper ml) and ruptured by three passages through a chilled French pressurecell at 135 MPa. 10 μl of 10 mM Tris/HCl, pH 7.5 was added per 200 μl ofpellet, followed by 20 μl of 150 mM DTT. DNase I (50 μg/ml) and RNase A(10 μg/ml) were added and incubated for 30 min at 37° C. Cell debris wasremoved by centrifugation (12000×g, 30 min, 4° C.). The supernatant wascentrifuged for 2 hours at 45,000 rpm at 4° C. The pellet wasresuspended in 5 ml high salt buffer (20 mM Tris-HCl, pH 7.0, 400 mMNH₄Cl, 10 mM MgAc₂, 6 mM β-mercaptoethanol) and layered onto a 7 ml17.5% sucrose cushion and centrifuged for 3 hours at 45,000 rpm at 4° C.The pellet was resuspended in a low Mg²⁺-buffer (10 mM Tris-HCl, pH 7.5,30 mM NH₄Cl, 0.3 mM MgCl₂, 6 mM beta-mercaptoethanol) and containedmostly intact ribosomes. MgCl₂ and acetic acid were added to finalconcentrations of 66 mM and 67% (v/v) respectively and the solution wasincubated on ice for 1 hour followed by centrifugation for 10 min. at10,000 rpm. The pellet was redissolved in the same solution and theextraction procedure was repeated once. The supernatants were combinedand dried in the speed-vac.

Before separating the ribosomal proteins by reversed-phase HPLC, therRNA was extracted. MgCl₂ and acetic acid were added to the ribosomes toa final concentration of 67 mM and 67% respectively and then incubatedon ice for 1 hour. The mixture was centrifuged at 10,000 rpm for 10 min.at 4° C. in a Sorvall SS-34 rotor. The procedure was repeated once morewith the pellet. The supernatants were combined and concentrated in thespeed-vac. The proteins were redissolved in 3% acetic acid, centrifugedat 10,000 rpm for 20 minutes at room temperature in an Eppendorffcentrifuge to remove any undissolved particles and injected onto apreparative reversed-phase HPLC system (L-6220 Intelligent Pump, L-4250UV-VIS Detector, Merck-Hitachi AG, Darmstadt, Germany). A gradient of10–25% B in 30 min., 25–35% B in 40 min., 35–36% B in 30 min., 36–40% Bin 40 min., 40–55% B in 60 min. and 55–90% B in 40 min. was run at 2mL/min. (A=0.1% TFA, B=80% acetonitrile/0.08% TFA), using a C₁₈preparative column (250×21 mm, Nucleosil 100—12 μm, Macherey-Nagel AG,Oensingen, Switzerland). The absorbance was measured at 220 nm.Fractions of 2 mL were collected and the amount of protein determined bythe method of Lowry and the proteins were identified by HPLC-MS/MSanalysis of tryptic digestions of the fractions.

Example 4 The Immobilised Membrane as a Flow-Through Chemical Reactor

Sequence Tagging: The chemistry of the method for sequence tagging wasas disclosed in Hoving, S., Münchbach, M., Quadroni, M., Staudenmann,W., and James, P. (2000) Anal. Chem. 72, 1006–1014. Multiple N-terminaltag generation from unseparated protein digests using a novel thioesterbased degradation reaction.

FIG. 2 a) shows schematically a section through part of an array 51 ofsubstantially identical flow-through chemical reactor cells 53 formed ina substrate 55, ands FIG. 2 b) shows a MALDI target slide and maskingplate as described below. The array 51 may comprise any suitable numberof cells, preferably arranged in a grid pattern comprising a number ofrows and columns, for example 5 rows and 10 columns, 8 rows and 12columns or 10 rows and 10 columns, etc. The construction of one suchcell 53 shown in FIG. 2 a) will now be described. Cell 53 is preferablyformed as a regularly shaped cavity 53, in this example a cube, formedin said substrate 55. The dimensions of the cavity are adapted to thedimensions of a capture means support e.g. a MALDI target slide 52similar to the MALDI target slide 12 described above but in thisinstance supporting a plurality, e.g. two, of capture means 67A and 67B.Capture means 67A and 67B are spatially separated and provided withsealing means (not shown) in order to prevent transferring of substancesfrom one capture target to the other. For the Tagging method it isenvisaged that two passageways exit from a gel-loading funnel similar tothat described in connection with FIG. 1 described above, one passagewayleading to capture means 67A and the other passage way leading tocapture means 67B. The protein is more or less equally divided betweenthe two capture means 67A and 67B. In the subsequent chemical taggingexperiment in the cell 53 a masking means such as an inert Teflonmasking plate 69, as shown in FIG. 2 b), provided with a though hole 68is positioned over the slide 52 such that one of each pair of capturemeans, e.g. capture means 67A is masked by the portion of a maskingmeans 69 which does not have a through hole, and does not undergodegradation whilst the other capture means 67B has the through hole 68positioned above it and therefore is able to be degraded. Thus an intactdigest is present which defines the parent peptides in the taggedposition allowing easier data extraction. FIG. 2 c) shows schematicallya view (not to scale) from above of a plate 75 with 100 loadingpositions 77 for stations of the type shown in FIG. 2 a); and;

FIG. 2 d) shows schematically a view (not to scale) from above of aplate 79 with 50 loading positions 81. The above mentioned embodimentsare intended to illustrate the present invention and are not intended tolimit the scope of protection claimed by the following claims.

1. A device for protein and/or peptide concentration in a sample, whichdevice comprises means for electroconcentrating (23) including a funnelshaped cavity (33) with a wide end and a narrow end; at least twoelectrodes (7, 35), one electrode (35) being positioned near to saidwide end and one electrode (7) being positioned nearer to said narrowend; one or more means for capturing proteins and/or peptides (17)located between said narrow end and said one electrode positioned nearsaid narrow end; and a size cut-off membrane (27) positioned above thecapture means (17) capable of separating non-digested proteins frompeptides, or larger size peptides from smaller size peptides.
 2. Thedevice of claim 1, wherein the capture means are adapted for use inMALDI-MS or MALDI-TOF.
 3. The device of claim 1, wherein the capturemeans (17) is an immobilization support capable of capturing positivelyand/or negatively charged peptides.
 4. The device of claim 1, whereinthe funnel shaped cavity (33) of the electroconcentration means (23) isin a position wherein said cavity is located in an essentially verticalposition, the wide end being in a higher position than the narrow end.5. A device for protein and/or peptide concentration in a sample, whichdevice comprises means for electroconcentrating (23) including a funnelshaped cavity (33) with a wide end and a narrow end; at least twoelectrodes (7, 35), one electrode (35) being positioned near to saidwide end and one electrode (7) being positioned nearer to said narrowend; two means for capturing proteins and/or peptides (17) locatedbetween said narrow end and said one electrode positioned near saidnarrow end: and said two capture means (67A, 67B) separated from eachother, one of which is preceded by a mask allowing unmodified peptidesto pass through and the other one of which allowing modified peptides topass through.
 6. The device of claim 5, wherein the two capture means(17) are immobilization support capable of capturing positively and/ornegatively charged peptides.
 7. The device of claim 5, wherein thefunnel shaped cavity (33) of the electroconcentration means (23) is in aposition wherein said cavity is located in an essentially verticalposition, the wide end being in a higher position than the narrow end.8. The device of claim 5, wherein the capture means are adapted for usein MALDI-MS or MALDI-TOF.